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Compositions and methods for biological production of isoprene

a biological and isoprene technology, applied in the direction of lyases, transferases, waste based fuel, etc., can solve the problems of inconvenient insufficient isoprene yields from naturally producing organisms, and limited industrial use of isoprene, so as to increase red pigmentation, increase red pigmentation, and increase the synthesis of isoprene precursors

Inactive Publication Date: 2016-01-21
CALYSTA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes non-naturally occurring methanotrophic bacteria that can convert a carbon feedstock into isoprene. These bacteria contain an exogenous nucleic acid encoding isoprene synthase, which is a key enzyme in the production of isoprene. The bacteria can be transformed with a variety of different nucleic acids to optimize isoprene production. The bacteria can be cultured in the presence of methane, methanol, natural gas, or unconventional natural gas as a carbon feedstock. The methods for producing isoprene involve culturing the bacteria and recovering the isoprene from the fermentation off-gas. The recovered isoprene can then be further modified into a dimer.

Problems solved by technology

Currently, isoprene's industrial use is limited by its tight supply.
Most synthetic rubbers are based on butadiene polymers, which is substantially more toxic than isoprene.
The isoprene yields from naturally producing organisms are not commercially attractive.
However, even with the use of relatively inexpensive cellulosic biomass as feedstock, more than half the mass of carbohydrate feedstocks is comprised of oxygen, which represents a significant limitation in conversion efficiency.
Isoprene and its derivatives (such as isoprenoids) have significantly lower oxygen content than the feedstocks, which limits the theoretical yield as oxygen must be eliminated as waste.
Thus, the economics of production of isoprene and its derivatives from carbohydrate feedstocks is prohibitively expensive.

Method used

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  • Compositions and methods for biological production of isoprene
  • Compositions and methods for biological production of isoprene
  • Compositions and methods for biological production of isoprene

Examples

Experimental program
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example 1

Cloning and Expression of Isoprene Synthase in Methanococcus Capsulatus Bath Strain

[0124]To create isoprene producing methanotrophic strains, a methanotroph expression vector containing a gene encoding isoprene synthase (IspS) was inserted into the Methylococcus capsulatus Bath, Methylosinus trichosporium OB3b, and Methylomonas sp. 16A via conjugative mating. An episomal expression plasmid (containing sequences encoding origin of replication, origin of transfer, drug resistance marker (kanamycin), and multiple cloning sites), was used to clone either a codon optimized Salix sp. IspS polynucleotide sequence (SEQ ID NO:19 for Methylococcus capsulatus Bath) downstream of a methanol dehydrogenase (MDH) promoter, or a Pueraria montana codon optimized IspS polynucleotide sequence (with the amino-terminal chloroplast targeting sequence removed) (SEQ ID NO:17 for Methylococcus capsulatus Bath) downstream of an IPTG-inducible (LacIq) promoter. Colonies of E. coli strain containing the IspS h...

example 2

Production of Isoprene by Methanococcus Capsulatus Bath Strain

[0126]Headspace gas samples (250 μl) from enclosed 5 mL cultures grown overnight of M. capsulatus Bath strain containing either a vector containing constitutive MDH promoter-Salix sp. IspS or a vector containing an IPTG-inducible (LacIq) promoter-Pueraria montana IspS (grown in the presence or absence of 0.1-10 mM IPTG) were obtained. Gas samples were injected onto a gas chromatograph with flame ionization detector (Hewlett Packard 5890). Chromatography conditions include an Agilent CP-PoraBOND U (25 m×0.32 mm i.d.) column, oven program 50° C., 1.5 min; 25° C., 1 min; 300° C., 10 min. The eluted peak was detected by flame ionization and integrated peaks were quantitated by comparison to isoprene standard (pure isoprene dissolved in deionized water).

[0127]M. capsulatus Bath produced more isoprene when expressing the Pueraria montana IspS as compared to expression of the Salix sp. IspS. In addition, and the amount of isopre...

example 3

Engineering a DXP Pathway with Improved Isoprene Production

[0128]Random mutations are introduced in the DXP pathway operon (i.e., DXS-DXR-IspD-IspE-IspF-IspG-IspH) for the purpose of generating novel gene sequences or regulatory elements within the pathway that overall, result in an improvement of enzymes for synthesis of the committed precursors of isoprene (IPP and DMAPP). To construct a facile high-throughput screening method for isolating an improved DXP pathway, a lycopene synthesis pathway comprising ggpps, crtB and crtI was utilized as a colorimetric reporter. A random mutagenesis library of the DXP pathway is created by error-prone PCR at low, medium, and high mutation rate using GENEMORPH® II random mutagenesis kit (Stratagene). The library is then cloned into a methanotrophic expression plasmid containing ggpps, crtB, and crtI gene sequences, whereby their polycistronic expression is driven by a strong methanotroph promoter sequence (e.g., methanol dehydrogenase promoter)....

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Abstract

The present disclosure provides compositions and methods for biologically producing isoprene using methanotrophic bacteria that utilize carbon feedstock, such as methane or natural gas.

Description

STATEMENT REGARDING SEQUENCE LISTING[0001]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 200206—411 WO_SEQUENCE_LISTING.txt. The text file is 58.5 KB, was created on Mar. 4, 2014, and is being submitted electronically via EFS-Web.BACKGROUND[0002]1. Technical Field[0003]The present disclosure provides compositions and methods for biologically producing isoprene, and more specifically, using methanotrophic bacteria to produce isoprene from carbon substrates, such as methane or natural gas.[0004]2. Description of the Related Art[0005]Isoprene, also known as 2-methyl-1,3-butadiene, is a volatile 5-carbon hydrocarbon. Isoprene is produced by a variety of organisms, including microbes, plants, and animal species (Kuzuyama, 2002, Biosci Biotechnol. Biochem. 66:1619-1627). There are two pathways for isoprene b...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P5/00C07C11/10C12N9/02C12N9/04C12N9/10C12N9/12C12N9/88
CPCC12P5/007C12Y406/01012C12N9/1022C12N9/0006C12N9/1205C12N9/0093C12N9/1241C07C11/10C12Y101/01267C12Y117/01002C12Y117/07001C12Y207/01148C12Y207/0706C12Y402/03027C12N9/88C12N15/52Y02E50/30
Inventor LEONARD, EFFENDIMINSHULL, JEREMYNESS, JON EDWARDPURCELL, THOMAS JOSEPH
Owner CALYSTA
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